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  • P H is a nonheme iron II

    2020-06-22

    P4H is a nonheme iron(II) dioxygenase that uses O2 and α-ketoglutarate as co-substrates (Fig. 1A). The three-dimensional structure of mammalian P4H is unknown. We reasoned that an electrophilic analog of α-ketoglutarate could serve as an irreversible inhibitor of the enzyme and, hence, a useful probe for active-site residues. Inspired by natural products such as trapoxin and epolactaene as well as artificial affinity labels, we designed 4-oxo-5,6-epoxyhexanoate (1) for this purpose. Our thought was that the epoxide oxygen would chelate to the active-site iron and thereby be activated for nucleophilic attack (Fig. 1B). We also noted that α-ketoglutarate and epoxy ketone 1 have the same number and a similar arrangement of nonhydrogen atoms. This attribute is important because of the inability of larger α-ketoglutarate analogs to inhibit catalysis by human P4H. Epoxy ketone 1, which also resembles 5-aminolevulinic acid, is a known competitive inhibitor of 5-aminolevulinic Ozagrel HCl sale dehydratase but does not alkylate that enzyme.
    Results and discussion
    Conclusions The need for small-molecule probes and inhibitors of P4H is evident from the lack of structural information about this essential enzyme as well as the list of fibrotic diseases associated with collagen overproduction. The newly discovered role of P4Hs in hypoxia25, 26, 27 and cancer, and the cardioprotection conferred by P4H inhibitors29, 30, 31 increase the imperative. Our identification of 4-oxo-5,6-epoxyhexanoate as an affinity label for P4H that is bioavailable upon esterification provides a means to address this need. On-going efforts in our laboratory are directed at identifying the alkylated enzymic residue, improving the potency of the epoxy ketone as an affinity label, and evaluating its selectivity towards other P4Hs.
    Experimental procedures
    Acknowledgments This Letter is dedicated to Professor Ashraf Brik on the occasion of his winning the 2013 Tetrahedron Young Investigator Award in Bioorganic & Medicinal Chemistry. We are grateful to Drs. Katrina H. Jensen and Lisa Friedman for guidance with chemical synthesis and in vivo assays, respectively. This work was supported by Grant R01 AR044276 (NIH). J.D.V. was supported by Molecular Biosciences Training Grant T32 GM007215 (NIH). E.A.K. was supported by Biotechnology Training Grant T32 GM083149 (NIH). The Micromass LCT® Mass Spectrometer was purchased with funds from Grant CHE-9974839 (NSF). This study made use of the National Magnetic Resonance Facility at Madison, which was supported by Grants P41 RR002301 and P41 GM066326 (NIH). Additional NMR equipment was purchased with funds from the University of Wisconsin, the NIH (P41 RR002781, P41 RR008438), the NSF (DMB-8415048, OIA-9977486, BIR-9214394), and the USDA.
    Introduction Around one third of the human world population, including a majority of children, is infected by parasitic nematodes [1,2]. In addition, plant-parasitic nematodes are one of the most infectious species in agriculture with an impact on economic loss of about 100 billion dollars per year [3]. The major barriers for drugs to penetrate parasitic nematodes are its collagenous cuticle, an exoskeleton, and an extracellular matrix (ECM). The free-living nematode C. elegans has been widely used as a surrogate model organism for parasitic nematodes [4], as well as for host-pathogen interactions [5], and other fundamental biological processes [6]. C. elegans is also used as a pioneering in-vivo model for biomedical research because about 40% of C. elegans genes are conserved in the human genome [7], and vice versa between 60 and 80% of human genes have a corresponding orthologue in the C. elegans genome [8]. In addition, 40% of human genes associated with diseases are well conserved in C. elegans [9]. C. elegans is genetically tractable for high throughput screens and is one of the best curated organisms for genetic, genomic, and phenotypic data. The vast array of openly shared molecular tools paved the way to gain molecular, functional, and mechanistic insights into gene and protein functions [8]. In particular, the two major extracellular matrices of C. elegans, the cuticle [10] and basement membrane [[11], [12], [13]], have recently become models to study cancer cell invasion [14] and aging [15]. However, a precise Gene Ontology term or a comprehensive compendium of genes predicted to form the C. elegans matrisome remains to be defined.